Az egyes fehérjesejt-integrinek élettani szerepének vizsgálata génhiányos (knockout) egerek segítségével = the physiological role of individual leukocyte integrins studies using genetificient (knockout) mice

A támogatott kutatások során a neutrofil granulociták működését vizsgáltuk genetikai, biokémiai és farmakológiai megközelítésekkel. Legfontosabb tudományos eredményeink alábbiak voltak: 1) A neutrofil granulociták integrinjei önmagukban nem képesek a sejtek teljes aktiválódását létrehozni (Jakus et al., J Immunol 2004; IF: 6.70) 2) A humán genom a korábban feltételezettnél lényegesen több ITAM-tartalmú molekulát kódol (Fodor et al., Immunol Lett 2006; IF: 2.14) 3) Az integrinek jelátvitele ITAM-függő mechanizmusokon keresztül jön létre (Mócsai et al., Nat Immunol 2006; IF: 27.60) 4) Egy új integrin-jelátviteli modell felállítása (Jakus et al., Trends Cell Biol 2007;IF: 12.43) 5) Egér neutrofilek immunkomplex-mediált aktiválódása az FcgammaRIII és az FcgammaRIV együttműködésével jön létre (Jakus et al., J Immunol 2008; IF: 6.29) Ezek az eredmények nagyban járulnak hozzá a neutrofilek működésének és az autoimmun gyulladásos folyamatok patomechanizmusának megértéséhez. | The sponsored research activity focused on the analysis of neutrophil functions by genetic, biochemical and pharmacological approaches. Our most important observations were the following: 1) Neutrophil integrins by themselves are not able to induce full activation of the cells (Jakus et al., J Immunol 2004; IF: 6.70) 2) The human genome contains significantly more ITAM-containing adapters than previousy thought (Fodor et al., Immunol Lett 2006; IF: 2.14) 3) Integrins utilize an ITAM-based signal transduction mechanism (Mócsai et al., Nat Immunol 2006; IF: 27.60) 4) Development of a new integrin signal transduction model (Jakus et al., Trends Cell Biol 2007;IF: 12.43) 5) Activation of murine neutrophils by immune complexes proceeds through a cooperative action of FcgammaRIII and FcgammaRIV (Jakus et al., J Immunol 2008; IF: 6.29) These results will strongly contribute to the understanding of neutrophil functions and the pathomechanism of autoimmune inflammatory diseases

Importance of Fc Receptor γ-Chain ITAM Tyrosines in Neutrophil Activation and in vivo Autoimmune Arthritis

Activating Fcγ receptors associated with Fc receptor γ-chain (FcRγ) are critical for mediating neutrophil effector functions in immune complex-mediated autoimmune diseases. FcRγ contains ITAM tyrosines and the in vivo role of these tyrosines has not been defined in neutrophils and arthritis. In this study, the in vivo functions of FcRγ ITAM tyrosines were characterized using wild type and ITAM tyrosine mutant (Y65F/Y76F) transgenic mice crossed to an FcRγ-deficient genetic background. FcRγ-deficient neutrophils showed undetectable cell surface expression of the activating Fcγ receptor IV, defective immune complex-induced superoxide production, degranulation and spreading. Although the re-expression of both the wild type and the ITAM tyrosine mutant (Y65F/Y76F) FcRγ could restore activating Fcγ receptor expression of FcRγ-deficient neutrophils, only the wild type transgenic form could mediate Fcγ receptor-dependent effector functions. In contrast, neutrophils carrying ITAM tyrosine mutant FcRγ were unable to produce superoxide, mediate degranulation and perform active spreading. In addition, our results confirmed the protection of FcRγ-deficient mice from autoimmune arthritis. Importantly, the presence of the wild type FcRγ transgene, in contrast to the ITAM tyrosine mutant transgene, partially reversed autoimmune arthritis development. The reversing effect of the wild type transgene was even more robust when animals carried the wild type transgene in a homozygous form. Collectively, FcRγ ITAM tyrosines play a critical role in the induction of neutrophil effector responses, the initiation and progression of an autoantibody-induced experimental arthritis in vivo, indicating a signaling, rather than just a receptor stabilizing function of the molecule

Platelets mediate lymphovenous hemostasis to maintain blood-lymphatic separation throughout life

Mammals transport blood through a high-pressure, closed vascular network and lymph through a low-pressure, open vascular network. These vascular networks connect at the lymphovenous (LV) junction, where lymph drains into blood and an LV valve (LVV) prevents backflow of blood into lymphatic vessels. Here we describe an essential role for platelets in preventing blood from entering the lymphatic system at the LV junction. Loss of CLEC2, a receptor that activates platelets in response to lymphatic endothelial cells, resulted in backfilling of the lymphatic network with blood from the thoracic duct (TD) in both neonatal and mature mice. Fibrin-containing platelet thrombi were observed at the LVV and in the terminal TD in wild-type mice, but not Clec2-deficient mice. Analysis of mice lacking LVVs or lymphatic valves revealed that platelet-mediated thrombus formation limits LV backflow under conditions of impaired valve function. Examination of mice lacking integrin-mediated platelet aggregation indicated that platelet aggregation stabilizes thrombi that form in the lymphatic vascular environment to prevent retrograde blood flow. Collectively, these studies unveil a newly recognized form of hemostasis that functions with the LVV to safeguard the lymphatic vascular network throughout life

Hematopoietic or osteoclast-specific deletion of Syk leads to increased bone mass in experimental mice

<p>Syk is a non-receptor tyrosine kinase critically involved in signaling by various immunoreceptors including B-cell-receptors and activating Fc-receptors. We have previously shown that Syk also mediates immunoreceptor-like signals required for the in vitro development and function of osteoclasts. However, the perinatal lethality of Syk^−/− mice precluded the analysis of the role of Syk in in vivo bone metabolism. To overcome that problem, we generated mice with osteoclast-specific (Syk^ΔOC) or hematopoietic (Syk^ΔHaemo) Syk deficiency by conditional deletion of Syk using Cre recombinase expressed under the control of the Ctsk or Vav1 promoter, respectively. Micro-CT analysis revealed increased bone trabecular density in both Syk^ΔOC and Syk^ΔHaemo mice, although hematopoietic Syk deficiency caused a more severe phenotype than osteoclast-specific Syk deficiency. Osteoclast-specific Syk deficiency reduced, whereas hematopoietic Syk deficiency completely blocked in vitro development of osteoclasts. Both interventions inhibited the resorptive activity of osteoclasts and osteoclast-specific gene expression. Kinetic analysis of Syk protein levels, Cre expression and the genomic deletion of the Syk^flox allele revealed complete and early deletion of Syk from Syk^ΔHaemo osteoclasts whereas Syk was incompletely deleted at a later stage of osteoclast development from Syk^ΔOC cultures. Those results provide an explanation for the in vivo and in vitro difference between the Syk^ΔOC and Syk^ΔHaemo mutant strains and suggest late activation of, and incomplete target gene deletion upon, osteoclast-specific Cre expression driven by the Ctsk promoter. Taken together, our results indicate that Syk plays an indispensable role in osteoclast-mediated in vivo bone resorption and suggest that Syk-specific inhibitors may provide therapeutic benefit in inflammatory and other diseases characterized by excessive osteoclast-mediated bone resorption.</p

Phosphoinositide 3-OH Kinase Regulates Integrin-Dependent Processes in Neutrophils by Signaling through Its Effector ARAP3

ARAP3, a GTPase activating protein for Rho and Arf family GTPases, is one of many phosphoinositide 3-OH kinase (PI3K) effectors. In this study, we investigate the regulatory input of PI3K upstream of ARAP3 by analyzing neutrophils from an ARAP3 pleckstrin homology (PH) domain point mutation knock-in mouse (R302, 303A), in which ARAP3 is uncoupled from activation by PI3K. ARAP3 PH domain point mutant neutrophils are characterized by disturbed responses linked to stimulation by either integrin ligands or immobilized immune complexes. These cells exhibit increased β2 integrin inside-out signaling (binding affinity and avidity), and our work suggests the disturbed responses to immobilized immune complexes are secondary to this. In vitro, neutrophil chemotaxis is affected in the mutant. In vivo, ARAP3 PH domain point mutant bone marrow chimeras exhibit reduced neutrophil recruitment to the peritoneum on induction of sterile peritonitis and also reduced inflammation in a model for rheumatoid arthritis. The current work suggests a dramatic regulatory input of PI3K into the regulation of β2 integrin activity, and processes dependent on this, by signaling through its effector ARAP3. Copyright © 2012 by The American Association of Immunologists, Inc

Lymphatic impairment leads to pulmonary tertiary lymphoid organ formation and alveolar damage

The lung is a specialized barrier organ that must tightly regulate interstitial fluid clearance and prevent infection in order to maintain effective gas exchange. Lymphatic vessels are important for these functions in other organs, but their roles in the lung have not been fully defined. In the present study, we addressed how the lymphatic vasculature participates in lung homeostasis. Studies using mice carrying a lymphatic reporter allele revealeded that, in contrast to other organs, lung lymphatic collecting vessels lack smooth muscle cells entirely, suggesting that forward lymph flow is highly dependent on movement and changes in pressure associated with respiration. Functional studies using CLEC2-deficient mice in which lymph flow is impaired due to loss of lympho-venous hemostasis or using inducible lung-specific ablation of lymphatic endothelial cells in a lung transplant model revealeded that loss of lymphatic function leads to an inflammatory state characterized by the formation of tertiary lymphoid organs (TLOs). In addition, impaired lymphatic flow in mice resulteds in hypoxia and features of lung injury that resemble emphysema. These findings reveal both a lung-specific mechanism of lymphatic physiology and a lung-specific consequence of lymphatic dysfunction that may contribute to chronic lung diseases that arise in association with TLO formation

Lymphatic mimicry in maternal endothelial cells promotes placental spiral artery remodeling

Molecular heterogeneity of endothelial cells underlies their highly-specialized functions during changing physiological conditions within diverse vascular beds. For example, placental spiral arteries (SAs) undergo remarkable remodeling to meet the ever-growing demands of the fetus-a process which is deficient in preeclampsia. The extent to which maternal endothelial cells coordinate with immune cells and pregnancy hormones to promote SA remodeling remains largely unknown. Here we found that remodeled SAs expressed the lymphatic markers PROX1, LYVE1, and VEGFR3, mimicking lymphatic identity. Uterine natural killer (uNK) cells, which are required for SA remodeling and secrete VEGFC, were both sufficient and necessary for VEGFR3 activation in vitro and in mice lacking uNK cells, respectively. Using Flt4Chy/+ mice with kinase inactive VEGFR3 and Vegfcfl/fl;Vav1-Cre mice, we demonstrated that SA remodeling required VEGFR3 signaling, and that disrupted maternal VEGFR3 signaling contributed to late-gestation fetal growth restriction. Collectively, we identified a novel instance of lymphatic mimicry by which maternal endothelial cells promote SA remodeling, furthering our understanding of the vascular heterogeneity employed for the mitigation of pregnancy complications such as fetal growth restriction and preeclampsia